Vesicular stomatitis virus (VSV), an animal pathogen, remains a paradigm of the non-segmented negative strand (NNS) RNA viruses to study the intricate molecular mechanisms of transcription and replication processes during the life-cycle of this class of viruses. The NNS RNA viruses constitute a vast multitude of pathogens, which inflict immense damage and destruction to humans, mammals, birds, fish and plants. The major human pathogens belonging to this group are rabies, measles, mumps, human parainfluenza, influenza, and many others, which still scourge the human race by their deadly infection. Recent emergence of hantaviruses and Ebola and Marburg viruses, also belonging to this family, has restimulated intense investigation to study NNS RNA viruses. Our laboratory has been actively engaged in examining the structure and function of the two key viral polypeptides of VSV;L (the large protein) and the phosphoprotein P (transcription factors), which together constitute the RNA polymerase complex. A major contribution in the last granting period to the field has been the firm establishment of the association of cellular proteins with the L protein expressed in insect cells, and the proposition of a new concept that the transcriptase and replicase are two distinct entities with different subunit compositions. In this renewal application, we have primarily focused on examining in detail the structure and function of both transcriptase and replicase of VSV purified from the infected mammalian cells. We are greatly inspired by our recent success in purifying for the first time the transcriptase complex to homogeneity and also the putative replicase from the infected cells and demonstrate that cellular heat shock protein, Hsp60, translation elongation factor, EF-1alpha and guanylytransferase, GT, are part of the transcriptase holoenzyme, whereas the replicase lacks the host proteins. Based on these findings, we have proposed three specific aims (1) to study in detail the functions of associated proteins of the transcriptase (2) to characterize the replicase and the replicative reactions and (3) to probe into the binding domain of the host protein in the L protein and association of host proteins in other NNS viruses. The knowledge gleaned from these studies will help provide valuable information with regard to the mechanism of gene expression of VSV and by extension to other NNS RNA viruses.